Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections, with biofilm formation playing a critical role in infection recurrence. The increasing prevalence of multidrug-resistant UPEC underscores the need for alternative strategies that target biofilms without imposing strong evolutionary pressure typical of conventional antibiotics This study identifies 2,2',4,4'-Tetrahydroxybenzophenone, also known as benzophenone-2 (BP-2), a UV filter widely used in sunscreens and cosmetic products as a promising candidate for repurposing as an antibiofilm agent against UPEC. BP-2 was evaluated for its antibiofilm activity against UPEC, as well as its ability to inhibit key virulence factors, including curli production, rugosity, and motility. Additionally, in vivo, in vitro, and in silico toxicity assessments were performed, along with 3D-QSAR analysis and molecular docking. BP-2 exhibited a minimum inhibitory concentration of 600 μg mL-1 and inhibited over 50% and 80% of UPEC biofilm formation at sub-inhibitory concentrations of 20 and 100 μg mL-1, respectively, without affecting bacterial growth or viability. Furthermore, BP-2 disrupted mature UPEC biofilms, impaired motility, rugose colony formation, and curli biogenesis, and showed antimicrobial activity against other pathogens. qRT-PCR demonstrated downregulation of curli biogenesis and motility related virulence genes while molecular docking suggested inhibition of the curli master regulator CsgD. 3D-QSAR analysis emphasized the role of hydroxyl substitutions at the 2nd and 4th positions of the benzophenone scaffold. BP-2 possesses potent antibiofilm activity against UPEC by targeting key virulence factors without affecting bacterial viability and has potential as a promising antibiofilm candidate for the treatment of UPEC-associated infections.
Precise characterization of Escherichia coli isolates plays a crucial role in the treatment and prevention of diseases in animal production. The classical diagnostic approach to define pathotypes of E. coli relies on detection of virulence genes. However, by targeting a limited set of genetic markers, routine PCR-based approaches may hinder the detection of atypical pathogenic isolates, especially in bacteria with high genomic plasticity. Genomic approaches were used to characterize a hybrid ETEC/ExPEC E. coli strain isolated from lambs submitted for necropsy over the course of six months, during an investigation of persistent neonatal mortality. Most lambs submitted for necropsy showed lesions suggestive of a septicemia. Routine PCR analysis detected genes encoding two ETEC-associated toxins but none of the ExPEC-associated virulence genes commonly targeted in routine diagnostics. Whole-genome sequencing revealed a diverse set of virulence genes, consistent with the hybrid nature of the strain. Genes encoding toxins were located on plasmids, while ExPEC-associated virulence factors were found to be chromosomally encoded. Genomic analyses revealed rapid antimicrobial resistance evolution, driven by plasmid acquisition. This case highlights how horizontal gene transfer promotes the development of hybrid pathotype and facilitates resistance genes acquisition, compromising traditional diagnostic approaches and treatment. ExPEC strains are particularly difficult to identify due to their diverse and ill-defined virulence markers, which are not always targeted by standard genotyping tests. Emergence of atypical E. coli strains such as those with hybrid pathotypes reinforces the need for more comprehensive methods such as whole-genome sequencing in veterinary diagnostics.
Mixing time is a key metric for characterizing bioreactors, as it governs bulk homogenization and correlates with the underlying flow field and shear exposure. Having knowledge of turbulent flow regimes is particularly important for shear-sensitive (mammalian) cells, which are often cultivated in single-use systems. To characterize such systems in terms of their mixing-time behavior, colorimetric pH-shift assays can be employed. However, operator-dependent variability during the reagent addition often limits the comparability of such experiments. To address this limitation and enhance reproducibility, a compact linear actuator was developed that enables automation and, thus, standardization of the addition process. The device was designed using computer-aided design software, fabricated via 3D printing, and subsequently integrated into mixing-time experiments. It is compatible with both stirred single-use bioreactors and glass bioreactors. For automated experiments using the linear actuator, neither the mixing time nor the replicate variance increased in the single-use bioreactor relative to the glass bioreactor under non-aerated conditions, demonstrating strong comparability between the two systems. The use of the linear actuator increased measurement precision, as evidenced by a 13.6% reduction in the coefficient of variation. This improvement resulted from the injection profiles, which were consistent in their immersion depth, immersion angle, tracer volume, and injection speed. Accordingly, this approach provides a low-cost, plug-and-play method to improve the reproducibility of mixing-time experiments, thereby facilitating bioreactor characterization, technology transfer, and validation of computational fluid dynamics models. KEY POINTS: Automated acid/base dosing improves reproducibility versus manual addition. Variation of the local mixing is detectable through spatially resolved analysis. Enables robust characterization of both stirred single-use and glass bioreactors.
Biofilms exhibit virulence traits that promote microbial protection, such as an extracellular matrix composed of exopolysaccharides (EPS), and contribute to host tissue damage via acid production. This study evaluated bacterial viability, biovolume, matrix (β-polysaccharides), and EPS (α-polysaccharides) formation in monospecies and mixed biofilms of Streptococcus mutans and Enterococcus faecalis, as well as pH and lactic acid production. Biofilms were grown on dentin blocks for 7 days, and aliquots were collected for colony-forming unit (CFU/mL) counts. Subsequently, biofilms were stained with LIVE/DEAD®, Calcofluor White®, and Alexa Fluor 647-dextran conjugate® to assess bacterial viability, matrix formation, and EPS formation, respectively, using confocal laser scanning microscopy (CLSM). Lactic acid production was determined by enzymatic spectrophotometry, and the pH of the culture medium was measured using a pH meter. Kruskal-Wallis and Dunn's tests were used for pH, viability, biovolume, matrix, and EPS analyses, while one-way ANOVA followed by Tukey's test was applied for CFU/mL counts and lactic acid production (α = 0.05). Dual-species biofilms showed higher viability and biovolume than S. mutans monospecies biofilms (p < 0.05), whereas the other parameters did not differ among groups (p > 0.05). Dual-species biofilms of S. mutans and E. faecalis may represent a suitable model for investigating antimicrobial strategies, as their association increased viability and biovolume, which are relevant features in cariology and endodontics.
This study aimed to evaluated the comprehensive effects of dietary Clostridium butyricumon the growth performance, digestion, immunity, and gut microbiota of Rana catesbiana larvae. Six groups (N0-N5), each with three replicates of 45 R. catesbeiana larvae, were established, using the dietary C. butyricum supplementation (0.25%-4%) (N1-N5) and an unsupplemented diet (N0) as control. The growth performance of bullfrog larvae in addition of 2% C. butyricum (N4) was the best. Regarding digestive function, the activities of intestinal lipase and chymotrypsin in the N4 group increased by 299% and 193% compared with the control group (P < 0.05), and the activity of liver amylase increased by 128%. In terms of immune regulation, the serum lysozyme and total nitric oxide synthase activities in the N4 group increased by 75% and 71% than the control group. The key antioxidant enzymes of superoxide dismutase and catalase, showed the highest activities were 46% and 57% higher than those of the control group. Intestinal histology showed that the muscle layer thickness and villus height of the N4 group increased by 22% and 16%. Microbial analysis revealed that the addition of C. butyricum significantly increased the alpha diversity index of gut microbiota. Two percent C. butyricum supplementation promotes bullfrog larval health through synergistic improvements in nutrient metabolism, immune defence, and intestinal microbiota homeostasis.
To investigate the antimicrobial resistance, β-lactamase genes, biofilm formation, and genomic characteristics of Klebsiella species isolated from diseased dogs in southeastern Brazil. Fifty-five Klebsiella isolates from dogs with clinical infections were identified by MALDI‒TOF MS. Antimicrobial susceptibility was assessed by disk diffusion. ESBL and carbapenemase genes were detected by PCR. Biofilm formation was evaluated using a microtiter plate assay. The only blaKPC-positive isolate underwent whole-genome sequencing and phylogenetic analysis with publicly available Brazilian genomes. K. pneumoniae was the predominant species (74.5%), followed by K. variicola (20%). Multidrug resistance was observed in 36.4% of the K. pneumoniae isolates. High resistance rates of the isolates were observed for fluoroquinolones, cephalosporins, and sulfonamides, whereas chloramphenicol showed moderate susceptibility. The most frequent β-lactamase genes were blaTEM-1 (34.5%) and blaCTX-M-15 (30.9%). A single isolate (1.8%) harbored blaKPC. No hypervirulence-associated genes were detected. All the isolates formed biofilms, predominantly with weak to moderate adhesion. Whole-genome sequencing of the blaKPC-positive isolate revealed K. pneumoniae ST11 (CG340) carrying multiple resistance genes, including blaKPC-2, and clustering with human-associated genomes within a high-risk lineage. Multidrug-resistant K. pneumoniae, including a high-risk ST11 blaKPC-2-producing clone, was identified in diseased dogs, supporting the need to investigate companion animals as possible reservoirs of clinically relevant antimicrobial resistance determinants. The combination of multidrug resistance and biofilm formation highlights the importance of continued surveillance and prudent antimicrobial use within One Health.
Antimicrobial resistance is a growing public health concern in India. It is driven largely by the misuse and overuse of antibiotics. This qualitative study aimed to examine how the Training and Communication (T and C) package influenced antibiotic prescribing practices of healthcare providers and prescription adherence among patients and caregivers at Civil Hospital, Manimajra, Chandigarh. A grounded theory approach was applied to qualitative data collected at baseline and endline. Purposive sampling was used to conduct focus group discussions and in-depth interviews with healthcare workers, patients, and caregivers. The topic guides were developed with reference to the capacity, opportunity, motivation-behavior framework. Data were transcribed, translated, manually coded, and thematically analyzed to identify drivers and barriers to prescription adherence before and after the intervention. Drivers of, and barriers to, prescription adherence included patient characteristics, doctor's workload, patient's knowledge about antibiotic uses, expectations for cheaper medicines, and the attitude of healthcare providers before the intervention. Based on these findings, a T and C package was developed for healthcare providers and patient communication. The use of rapid diagnostic tests and prescription communication helped in early diagnosis and increased patient's adherence to the prescribed medicine after the intervention. Patients in the intervention arm reported supportive engagement with the healthcare professionals. The affordability of medicine was a major barrier to prescription adherence across arms, pre- and postintervention. Training of service providers and communication with patients were important for ensuring prescription adherence and optimal use of antibiotics. The affordability of medicines was the biggest challenge.
This study aimed to formulate and characterize a novel next-generation nutraceutical combining a mixture of Limosilactobacillus fermentum strains (139, 263, and 296) candidates for probiotic use and freeze-dried Pilosocereus pachycladus (F. Ritter) (facheiro) cladodes, focusing on its physical, physicochemical, and phytochemical properties, the survival and viability of L. fermentum, and the stability and performance of the nutraceutical under simulated gastrointestinal digestion over 90 days of controlled storage (13% relative humidity; 4 and 25°C). The nutraceutical had high contents of insoluble and soluble fibers, as well as antioxidant phenolic compounds and flavonoids, which supported the survival of L. fermentum strains during freeze-drying (>90%) and conferred protection under gastrointestinal conditions, with survival rates exceeding 50%. Among the storage conditions, refrigerated storage provided superior long-term stability, preserving bioactive compounds and the viability of L. fermentum cells. Simulated gastrointestinal digestion demonstrated effective delivery of antioxidant compounds and viable L. fermentum cells, even in the presence of low pH, digestive enzymes, and bile salts. The combination of L. fermentum strains candidates for probiotic use and freeze-dried facheiro cladodes demonstrated technological stability and functional performance, highlighting facheiro cactus as a valuable bioresource for probiotic delivery. Further studies using advanced in vitro models, such as colonic fermentation, and preclinical trials are warranted to substantiate the health benefits of this nutraceutical.
Ultraviolet C radiation (UVC; λ = 100-280 nm) is a well-established physical agent for microbial inactivation, widely applied in surface and environmental disinfection. Considering the increasing demand for efficient, safe, and automated disinfection systems, this study compares the performance of two low-pressure mercury lamps with different wattages and a novel UVC-LED array in terms of UVC irradiance, DNA damage induction, and microbiological inactivation. DNA photodamage was assessed in vitro via cyclobutane pyrimidine dimer (CPD) detection, and bacteriological inactivation was evaluated with four relevant pathogenic bacteria (Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae carbapenemase (KPC), and Pseudomonas aeruginosa) exposed to increasing UVC doses (0-50 J/m²). All UVC sources induced a dose-dependent increase of DNA lesions and microbial inactivation, with 50 J/m² achieving an average inactivation rate of 99.98%. Although higher-power mercury lamps delivered higher dose rates (J/m²/s), the UVC-LED device showed comparable germicidal efficacy, with advantages in energy efficiency, operational lifespan and environmental safety. The results presented in this work indicate the feasibility of complementing the use of mercury-based lamps with UVC-LEDs due to its great potential application for point-of-use disinfection approaches. Furthermore, the obtained results also highlight the importance of controlled digital dosimetry to improve the UVC-based technologies application for disinfection of bacteria of hospital concern.
The aim of this study was to examine the fate of Escherichia coli strain 0611, used as model for a water-borne bacterial contaminant, in leafy green production systems. Irrigation water from two different sources were inoculated with the strain and applied via overhead irrigation to endive and spinach plants grown under open-field conditions. Inoculum levels were 10³ CFU ml⁻¹, representing occasional water contamination, and a 1,000-fold higher density to enable detailed quantification across plant compartments. Three experiments assessed bacterial persistence after single or double irrigation events at varying intervals, and under continuous irrigation. Colony-forming units were measured on leaves and in rhizosphere soils. Under continuous irrigation with the low inoculum dose, E. coli 0611 was only sporadically detected on leaves. At the higher dose, the interval between irrigation and harvest had a greater impact on bacterial presence than water source. Decline patterns on leaves and in endive rhizosphere soil followed double-exponential models. Rhizosphere soil represents an additional risk factor for transmission of human pathogens to fresh produce. Although bacterial populations declined rapidly on leaves, they did not completely disappear from rhizosphere soil. A preharvest interval of at least four days after the final irrigation substantially reduces the risk of pathogen transfer to fresh produce. The experimental data support evidence-based recommendations for producers to reduce the risk of microbial contamination of fresh produce, thereby contributing to safer production of ready-to-eat vegetables.
The rising wave of antimicrobial resistant organisms, together with the lack of novel group of antibacterial agents, represents a major concern in modern medicine. This study aims to investigate the anti-staphylococcal potential of two semi-synthetic derivatives of naturally occurring nitrogen-containing molecules [2-O-(3-methoxybenzoyl)-3-O-methylpancracine (1 nm) and 2-O-(3,5-dimethoxybenzoyl)-3-O-methylpancracine (2 nm)]. The two semi-synthetic analogues were evaluated against reference bacterial strains and clinical bacterial isolates. Furthermore, the most active compound, 1 nm, was subjected to synergistic activity screening with the commercial antibiotics against MRSA. The toxicity profile of 1 nm was further explored using in vitro (HepG2 and HK-2 cells) and in vivo (Galleria mellonella) models, accompanied by an insight into its in silico physicochemical characteristics and cytotoxicity. Our results suggest that this derivative possesses promising activity against clinically relevant staphylococci, while showing a favourable safety and bioavailability profile. In addition, this compound showed synergistic or additive effects in combination with several clinically important antibiotics. 1 nm demonstrates promising adjuvant anti-staphylococcal activity, with a favourable safety profile and an additive interaction with the last-resort antibiotic linezolid, supporting its potential to mitigate resistance development within combination strategies.
Fumigation of biological safety cabinets (BSCs) is essential for their safe use, maintenance, and quality assurance of work performed. Hydrogen peroxide (HP) is frequently used as an alternative fumigant to formaldehyde, applied either at high concentrations (>30%) in vapour phase, or low concentrations (5%-12%) as an aerosol (aHP). There is, however, limited evidence on the efficacy of low-concentration aerosolized HP (aHP) fumigation to inactivate microorganisms within BSCs. This study evaluates the antimicrobial efficacy of aHP within a BSC III, comparing fumigation cycle parameters against a range of microorganisms. Biological indicators (BIs) were prepared by inoculating stainless steel coupons with either Geobacillus stearothermophilus spores, methicillin-resistant Staphylococcus aureus (MRSA), or Mycobacterium fortuitum. BIs were placed within a BSC III and fumigated with one or six diffusion cycles of aHP, with one diffusion cycle recommended for routine decontamination and six diffusion cycles advised for pre-maintenance decontamination, providing increased aHP quantity and exposure time. Fumigation with one aHP diffusion achieved a > 6 log10 reduction in G. stearothermophilus spore CFU, with 2.24 and 0.46 log10 average reductions for MRSA and M. fortuitum, respectively. Six-diffusion fumigation achieved significantly reduced CFU counts for all microorganisms (average log10 reductions of 7.05, 5.25, and 5.26 for G. stearothermophilus, MRSA, and M. fortuitum, respectively). Incomplete BI inactivation was, however, observed for all microorganisms. Increased aHP diffusion cycles enhanced microbial reductions; however, complete elimination of microorganisms was not observed. BIs, which represent likely contaminated microorganisms should be used when validating fumigation effectiveness.
In recent years, porcine circovirus type 2 (PCV2) has emerged as one of the most economically significant viral pathogens affecting the global swine industry, leading to substantial economic losses. Accumulating evidence indicates that oxidative stress facilitates PCV2 infection and replication, whereas alleviating oxidative stress can attenuate this process. Notably, glutathione and selenium play pivotal roles in the antioxidant response. Accordingly, we selected a glutathione-producing strain of Bacillus pumilus for selenium enrichment and investigated its inhibitory effects on PCV2 replication. This study aims to provide experimental evidence for the development of microecological preparations with antiviral activity. In this study, microbial colony counting, analysis of the biological characteristics of bacteria, histopathological analysis, biochemical indicator analysis, quantitative real-time PCR, and Western blotting were used to evaluate the effects of the selenium-enriched Bacillus pumilus composite probiotic on PCV2 replication in a mouse model. The results demonstrated that this Bacillus pumilus strain exhibited high glutathione production, favorable biological characteristics, and strong selenium-enriching capacity. The composite probiotic preparation modulated the JAK/STAT signaling pathway and elevated the phosphorylation levels of JAK1, STAT1, and STAT2. This further upregulated the expression of key antiviral effector genes Mx1 and Oas1 in the mouse spleen, thereby inhibiting PCV2 replication in vivo. In this study, selenium-enriched, glutathione-producing Bacillus pumilus exhibits antiviral potential. It suppresses in vivo PCV2 replication via alleviating oxidative stress, which provides the theoretical basis and experimental support for the development of novel antiviral microecological preparations.
Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP), which possesses both high pathogenicity and drug resistance, has become a major clinical challenge. The role of AcrAB-TolC efflux pump in its resistance mechanism has attracted increasing attention. This study aimed to clarify the role of the AcrAB-TolC efflux pump in carbapenem resistance of CR-hvKP, confirm the role of AcrB in the function of the AcrAB-TolC efflux pump, and reveal the regulatory mechanism of RamA and RamR. This study provides a theoretical basis for the treatment and prevention of CR-hvKP infections. Thirty-eight non-repetitive clinical CR-hvKP isolates were analyzed. Minimum inhibitory concentrations (MICs) were determined using broth microdilution. Efflux pump activity was evaluated by comparing MICs before and after addition of CCCP. The Carbapenemase Nordmann-Poirel (Carb NP) test excluded carbapenemase-mediated resistance. PCR was used to detect virulence (iucA, rmpA, rmpA2, iroB, Peg344) and efflux pump (acrA, acrB, tolC) genes. Bioinformatics tools (KaKs_Calculator and Datamonkey) and RT-qPCR identified and quantified acrB expression. Gene knockout and overexpression, combined with MIC testing and a Galleria mellonella infection model, assessed effects on resistance and virulence. RT-qPCR evaluated RamA and RamR expression. acrB was identified as a major contributor to efflux-mediated resistance. Its expression decreased by 65% in the ramA knockout strain and increased by 40% in the ramR knockout strain. AcrB-positive strains significantly reduced larval survival. AcrB appears to be a major component of the AcrAB-TolC efflux system contributing to carbapenem resistance and potentially influencing virulence in CR-hvKP. RamA and RamR significantly regulate AcrB expression and modulate efflux-associated resistance.
Leuconostoc lactis is a heterofermentative lactic acid bacterium widely used as a starter culture in fermented foods. The L. lactis B34-1-7-1 strain, isolated from Panax ginseng sprouts cultivated under white LED light, has demonstrated promising probiotic potential, including antioxidant, anti-inflammatory, and anti-cancer activities, in previous studies. The genome of L. lactis B34-1-7-1 was sequenced and analyzed to evaluate its probiotic potential and safety for industrial applications, focusing on its genomic features and functional characteristics. The genome of L. lactis B34-1-7-1 was sequenced using a hybrid Illumina-PacBio approach. The complete genome is composed of a circular chromosome (1,648,351 bp) and a plasmid (12,481 bp), with an overall guanine-cytosine (GC) content of 43.6%. The genome encodes 1,661 coding sequences (CDSs), 68 transfer RNAs, and 12 ribosomal RNAs. Functional classification assigned 99.15% (1,647 genes) of the CDSs to Clusters of Orthologous Groups (COGs). Genome-based screening identified no known antibiotic resistance genes or virulence factors, supporting the safety of this strain. In addition, 23 genes associated with reductase and oxidase activities were identified, providing a genetic basis for its observed antioxidant potential. These results provide a foundational genomic resource supporting the application of L. lactis B34-1-7-1 in the functional food and probiotic industries.
This study aimed to evaluate whether combining phages with different properties enhances antibacterial efficacy against individual strains and enables the simultaneous control of multiple bacterial species belonging to different genera. Our phage collection was screened for phages active against Aeromonas hydrophila, Escherichia coli, Salmonella enterica Typhimurium and Vibrio parahaemolyticus. Four phages were selected: AH-1, ECA2, phSE-5 and vB_VpS_LMAVpSH, originally isolated using A. hydrophila, S. Typhimurium, E. coli and V. parahaemolyticus as hosts, respectively. An initial phage cocktail (CK3) was prepared excluding phage SH, which was later included in a modified cocktail (CK4). Phage efficacy was evaluated in vitro (in nutrient-rich media and synthetic seawater) against bacterial strains individually and against mixed cultures of the four bacteria, for 12 h at 25°C. Both cocktails were associated with greater bacterial reduction compared with individual phage treatments, alongside reduced bacterial regrowth under the tested conditions. Although efficacy decreased in nutrient-limited conditions, phages still inhibited bacterial growth. The findings of this study provide experimental evidence of phage-bacteria interactions in multispecies systems and offer proof-of-concept evidence supporting further evaluation of phage cocktail approaches under aquaculture-relevant conditions.
Determining the mixing time in a mixing apparatus enables the evaluation of mixing quality and, therefore, represents a valuable tool for characterizing unit operations in process engineering. Mixing is highly relevant in both upstream processes (e.g., bioreactors) and downstream processes (e.g., blending tanks in diafiltration) in biotechnology and pharmaceutics. The aim of combining a colorimetric method with video capture and automated image analysis is to provide a robust, standardized methodology for determining mixing times in transparent bioreactors, such as laboratory-scale glass bioreactors and single-use bioreactors. A Python-based tool for video analysis was developed for this purpose. The Python script provides different mixing indices that enable the evaluation of both global and local mixing times under varying stirring speeds and aeration rates. This approach offers deep insights into the mixing process and enables the identification of heterogeneities. A round-robin study involving members of the DECHEMA Working Group "Single-Use Technologies for Bio-Based Applications" is currently being conducted to enhance the analysis and ensure reproducibility across different laboratories, thereby providing a robust basis for validating computational-fluid dynamics simulations. Initial reproducibility issues were identified and addressed in the standard operating procedure, demonstrating that a framework for automated analysis and improved standardization has been established. KEY POINTS: • Mixing-time experiments conducted at different aeration rates and stirring speeds • Automated video analysis for determining mixing times on both global and local scales • Identification of heterogeneities through visualization of local mixing times.
Multidrug-resistant Citrobacter freundii, particularly isolates carrying extended-spectrum β-lactamases and carbapenemases are increasingly difficult to treat in clinical settings. To explore alternatives to antibiotics, we isolated a novel lytic bacteriophage from hospital wastewater in Zhengzhou and designated it vB_Cf_HW01. TEM revealed a siphovirus with a 50 nm capsid and 160 nm tail. Genomic analysis identified vB_Cf_HW01 as a novel Drexlerviridae species. Our results indicated an optimal MOI of 0.001, defined as the initial infection ratio that yielded the highest progeny titer. The lysis curve showed that vB_Cf_HW01 effectively inhibited host cell proliferation at this low MOI. Adsorption kinetics and one-step growth assays showed 97.17% adsorption within 14 min, a 10 min latent period, and a burst size of 256 PFU cell-1. The phage remained stable from -20 to 60°C and pH 4 to 10, resisted chloroform exposure, and retained residual infectivity after 90 min of UV irradiation. Sequencing revealed a 49 563 bp linear dsDNA genome (Accession: PX694430.1) containing 94 open reading frames. Genomic analysis identified no known virulence or antibiotic resistance genes. CLSM with live/dead staining showed severe disruption of mature C. freundii biofilms after 12 h of vB_Cf_HW01 treatment. Live cell coverage decreased from 9.17 ± 0.70% to 3.03 ± 0.92%, whereas dead cells increased from 0.14 ± 0.01% to 7.87 ± 0.18%. In an in vivo Galleria mellonella infection model, a single phage dose (MOI 10) rescued 36.67% of infected larvae. Thus, vB_Cf_HW01 is a promising candidate against drug-resistant C. freundii infections.
Orientia tsutsugamushi strain Ikeda is a scrub typhus reference strain originally described in Japan, and Ikeda 56-kDa type-specific antigen sequence types have also been reported in South Korea. However, complete genome resources for South Korean Ikeda-genotype isolates remain limited. Here, we generated complete genomes for two archived clinical O. tsutsugamushi isolates from northern South Korea, CH219 and K4-135, using a PacBio HiFi and Illumina hybrid assembly approach and compared them with the Japanese reference strain Ikeda and the South Korean reference strain Boryong. Both genomes were assembled as single circular chromosomes and contained a substantial fraction of duplicated identical coding sequences, consistent with the highly repetitive nature of O. tsutsugamushi genomes. Strains CH219 and K4-135 had identical 56-kDa TSA sequences and MLST profiles to those of the Ikeda reference strain and clustered within the Ikeda-associated clade in recombination-filtered core-genome phylogeny and ANI analyses. Within this comparison, the two South Korean isolates showed more closely related to each other than to the Japanese Ikeda reference genome. Whole-genome dot plots further indicated structural variation among the Ikeda-associated genomes. Insertion sequence (IS) profiling showed differences in IS-related CDS copy number patterns between the Boryong reference genome and the Ikeda-associated genomes, with strain Boryong showing a higher observed number of ISOt6-related CDS hits and fewer high-identity ISOt3-related hits under the applied thresholds. Together, these genomes provide new resources for Ikeda-like O. tsutsugamushi strains detected in South Korea and support the need for expanded complete and well-supported whole-genome data to better understand genome diversity in scrub typhus agents.
To explore healthcare professionals' (HCPs) experiences of diagnosing cellulitis, identify unmet diagnostic needs and determine the desirable characteristics of an ideal diagnostic test. Qualitative study using semistructured interviews, analysed thematically. Community and secondary care services across the UK from January 2023 to September 2023. 25 UK-based HCPs (eg. nurses, paramedics, pharmacists, general practitioners and hospital physicians) with recent experience of managing cellulitis. Inclusion required recent involvement in cellulitis care; no specific exclusion criteria were applied. All participants completed the interview. Four major themes were identified. Theme 1 described patient and disease characteristics that make diagnosis difficult, including atypical or recurrent presentations, high-risk patients, pre-existing wounds or ulcers, darker skin tones and prior antibiotic use, which represented specific use cases in which a test could be deployed. Theme 2 highlighted clinician and patient behaviours contributing to misdiagnosis, including reliance on cellulitis as the 'default' diagnosis, limited dermatology/tissue viability knowledge and access, risks of not treating and patient expectations. Theme 3 examined candidate diagnostic aids (evaluated in early studies) and ideas for novel diagnostic tools, noting that current tools are insufficiently specific, while enthusiasm for new technologies was countered by concerns about usability, comfort, infection control and training needs. Theme 4 identified key characteristics of an ideal test: portable, rapid, equitable, usable across community and hospital settings and providing a probabilistic result rather than a binary outcome. Cellulitis remains diagnostically challenging across multiple care settings. Developing tools that can distinguish patients who genuinely require antibiotic therapy from those with non-infective inflammation would be particularly valuable. The greatest potential for impact lies in improving diagnostic specificity to reduce misdiagnosis, unnecessary antibiotic use and avoidable healthcare attendance and admission. Broad engagement of stakeholders will be essential to define acceptable performance parameters and guide novel technology development.